Hook Adaptors Induce Unidirectional Processive Motility by Enhancing the Dynein-Dynactin Interaction

Mara A Olenick,Mariko Tokito,Malgorzata Boczkowska,Roberto Dominguez,Erika L.F Holzbaur

doi:10.1074/jbc.m116.738211

Abstract

Cytoplasmic dynein drives the majority of minus end-directed vesicular and organelle motility in the cell. However, it remains unclear how dynein is spatially and temporally regulated given the variety of cargo that must be properly localized to maintain cellular function. Recent work has suggested that adaptor proteins provide a mechanism for cargo-specific regulation of motors. Of particular interest, studies in fungal systems have implicated Hook proteins in the regulation of microtubule motors. Here we investigate the role of mammalian Hook proteins, Hook1 and Hook3, as potential motor adaptors. We used optogenetic approaches to specifically recruit Hook proteins to organelles and observed rapid transport of peroxisomes to the perinuclear region of the cell. This rapid and efficient translocation of peroxisomes to microtubule minus ends indicates that mammalian Hook proteins activate dynein rather than kinesin motors. Biochemical studies indicate that Hook proteins interact with both dynein and dynactin, stabilizing the formation of a supramolecular complex. Complex formation requires the N-terminal domain of Hook proteins, which resembles the calponin-homology domain of end-binding (EB) proteins but cannot bind directly to microtubules. Single-molecule motility assays using total internal reflection fluorescence microscopy indicate that both Hook1 and Hook3 effectively activate cytoplasmic dynein, inducing longer run lengths and higher velocities than the previously characterized dynein activator bicaudal D2 (BICD2). Together, these results suggest that dynein adaptors can differentially regulate dynein to allow for organelle-specific tuning of the motor for precise intracellular trafficking.

Highlights

Microtubules provide a polarized highway to facilitate the transport of organelles and vesicles throughout the cell
This polarity ensures that microtubule motors drive motility in a specific direction; kinesin motors generally drive plus end motility, whereas minus end traffic is primarily driven by cytoplasmic dynein
We use the dimerizer cTMP-Htag, a small molecule made of a HaloTag proteins (Halo) tag (Htag) ligand linked to a photocaged trimethoprim (TMP)

Summary

Introduction

Microtubules provide a polarized highway to facilitate the transport of organelles and vesicles throughout the cell. The organelle redistribution induced by either of the Hook proteins was similar to that observed upon recruitment of the known dynein activator BICD2, suggesting that Hook proteins act as dynein adaptors (Fig. 1, B and C). With the expression of Hook proteins, we observed an increase in the co-precipitation of dynein by the anti-p150Glued antibody compared with a control experiment in which only the Halo tag was expressed (Fig. 3B).

Results

Conclusion